1. Purification strategies By : Miss Thida Chanyachukul AIDs Production group

2. Outline  General concepts in Purification strategies  Principle of purification operations  Special considerations in our biopharmaceutical products  Ritonavir (Protease inhibitor)  Vaccine  IL-2  Conclusions  References

3. General concepts Purification strategies  important prerequisite in pharmaceutical manufacture  more predictable and controllable  not all problems in purification system are solved by the acquisition of sophisticated laboratory equipment and column packing  difficult to find optimum conditions of choosing suitable methods

4. General concepts  base the purification of biopharmaceutical product on knowledge on structure / function / particular structural details  Conversely, results from the application of a particular purification method can often be interpreted in terms of molecular properties

5. General concepts  Production can be divided into  upstream processing the initial fermentation process, which results in the initial generation of product.  downstream processing the actual purification of the product and generation of finished product format followed by sealing of the final product containers

6. Categories of unwanted  Components present due to process conditions :  Host-cell-derived components  Process-derived components  Components present due to contamination :  Adventitious agent

7. In Plant-scale working  volumes of between 2-5 liters  the most useful methods of purification  recrystallization for solids  distillation or steam distillation for liquids  chromatography should be avoided  but if it is necessary, then medium pressure liquid chromatography (mplc) is the method of choice

8. Principle of purification operations Filtration Chromatography Distillation Crystallization or Recrystallization

9. Basic concept of Filtration  Technique : pass the solution, cold or hot, through a fluted filter paper in a conical funnel removes particulate impurities from liquids or collect insoluble or crystalline solids from solution  the solid particles are too fine centrifugation should be used

10. Basic concept of Gel filtration  different amount of time different solute stay within the liquid phase that is entrapped by the matrix  pore dimension  gel structure  solute size  uncomplicated / straightforward technique

11. Industrial filtration devices

12. Basic concept of chromatography  = a group of separation techniques, which are characterized by a distribution of the molecules to be separated between two phases, one stationary and the other mobile phase  molecules with a high tendency to stay in the stationary phase will move through the system at a lower velocity than will those which favor the mobile phase.  the shape, rigidity and particle size distribution profile of the gel matrix are important parameters

13. Basis chromatography  Ion exchange differences in protein surface charge at a given pH  Gel filtration differences in size/shape of different protein  Hydrophobic interaction chromatography differences in the size and extent of hydrophobic patches on the surface of proteins  Affinity chromatography ability of a protein to bind in a bio- specific manner to a Chosen immobilized ligand

14. mplc  simplified  cheaper  easy to predict which fractions will contain each component  silica / alumina / ion exchange resin,  the appropriate size of column and the correct solvent  linked to a fraction collector / An UV / refractive index detector / TLC

15. Basic concept of Ion exchange chromatography (IEC)  the more highly charged a protein the more strongly it will bind to a given, oppositely charged ion exchanger  the more highly charged ion exchangers bind proteins more effectively than weakly charged

16. Advantages of Ion exchange chromatography (IEC)  versatility  high resolving power  high loading capacity  multiple inlets  column with large diameter  straight forward basic principle.

17. Basic concept of affinity chromatography  = the exploitation of various biological affinities for adsorption to a solid phase the ligand immobilized on the solid phase the counterligandpassing the chromatographic column  stages : adsorption, washing, elution and column regeneration

18. Biological interactions used in affinity chromatography Ligand Counterligand Antibody antigen, virus, cell Enzyme substrate analogue, inhibitor, co-factor Lectin polysaccharide, glycoprotein, cell surface receptor, cell Nucleic acid nucleic acid-binding protein (enzyme or histone) Hormone, vitamin receptor, carrier protein Sugar lectin, enzyme or other sugar binding protein

19. Special consideration on Affinity chromatography  association strength, between ligand and counterligand if it is too weak no adsorption if it is too strong difficult to elute the protein adsorbed  pH  salt concentration  dissociation substances  appropriate specificity affecting the protein to be isolated

20. Basic concept of distillation  The distillation process involves  boiling a liquid  condensing the vapors  the resulting liquid  suitable for all organic liquids and most of the low- melting organic solids  the efficiency depends on the difference in boiling points of the pure material and its impurities

21. Steps of crystallization or recrystallization  The impure material  dissolved at or near the boiling point to form a near-saturated solution.  the hot solution is filtered to remove any insoluble particles  allowed to cool  fast cooling generate many nuclei of small crystals  the dissolved substance crystallizes out  centrifuge or filter crystals from mother liquor  washed free of mother liquor with a little fresh cold solvent  dried

22. Basic concept of crystallization or recrystallization  only as a last step  yield of about 90% at best  10% yield loss is quite high  purified with additional processing which are much more quantitative such as extraction or chromatography

23. Special considerations in our Biopharmaceutical products Vaccine / IL-2 Protein RitonavirOrganic molecule

24. Plant-scale process of protein Working cell bank vial removed from storage propagation of working bank cells, generating starting cultures cell harvesting and recovery of crude protein production-scale cell culture concentration (if necessary) and initial purification main purification (chromatography) product filling, freezing and sealing final product formulation labeling and packaging

25. Upstream purification of Ritonavir + chromatography 2-aminoaldehyde diols (white solid) bromoacetate (white solid) pricipitation filtration chromatography filtration diamine (white solid) compound X (white solid) epoxide (white solid) distillation chromatography resin compound compound XXXIIIa Ritonavir

26. Downstream purification of Ritonavir Hydrophobic interaction Affinity chromatography chromatography dryer final product formulation

27. Conclusion  purity is a matter of degree  sufficient pure for some intended purpose  absolute purity is an ideal which can never be shown to be attained  the starting material should be of the grade commercially available  In general, at least two different methods, such as recrystallization and distillation, should be used in order to ensure maximum purification  the decision to market the product in liquid or powder form must be determined experimentally, as there is no way to predict the outcome for any particular material

28. References  Berthold, W. and Walter, J. Protein purification: aspects of processes for pharmaceutical products. Biologicals 1994;22:135- 150.  Janson JC. And Ryden L. Protein purification; principles, high- resolution methods, and application: VCH publishers, Inc. 1989.  Hesse F. and Wagner R. Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures. Trends in Biotechnology. 2000 ;18(4):173-180  WHO study group. Acceptability of cell substrates for production of biologicals. WHO technical report series; 1987: 747, 1-29.  Walter, J and Allgaier, H. Validation of downstream processes. In: Mammalian Cell Biotechnology in Protein Product.1997; 453-48.  Perrin DD., Armarego WLF. and Perrin DR. Purification of laboratory Chemicals. 2 nd ed. : Pergamon Press. 1980.  White HL. Introduction to industrial chemistry : John Wiley & Sons, Inc. 1986

29. Thank you